Characterization of Arthrospira / Spirulina strains: Molecular Aspects

Report (1999)

We present the results of a phylogenetic study, based on ARDRA of the rDNA operon, of 38 Arthrospira (‘Spirulina’) cultivated clonal strains from four continents. In addition, duplicates from different ... [more ▼]

We present the results of a phylogenetic study, based on ARDRA of the rDNA operon, of 38 Arthrospira (‘Spirulina’) cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 54 tested cultures. Three living samples from Earthrise Farms ponds (September 1997), four freeze-dried samples from EF ponds (August 1996, February and March 1997) and a powder of ‘Spirulina pacifica’ were also included in the study. The strain Spirulina laxissima SAG 256.80 was used as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by ARDRA, and thus the Internal Transcribed Spacer (ITS) was selected as a molecular taxonomic marker. The ITS sequences situated between the 16S and the 23S rRNA genes were amplified by PCR and yielded amplicons of about 540 bp. The amplicons were digested with four restriction enzymes (EcoR V, Hha I, Hinf I, Mse I) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters (Clusters I and II), of which Cluster I was divided into Subclusters I.A and I.B. Four freeze-dried samples from EF cultivation ponds (Summer 1996 and Winter 1997), as well as a sample of powder sent as ‘Spirulina pacifica’ appeared to contain a mixture of genotypes from Clusters I and II. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology. Direct use of cells for PCR seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of BSA (Bovine Serum Albumin) in the PCR mix. In order to study in more depth the genotypic relationships of Arthrospira, we have obtained the ITS sequence of 19 cultures and 7 samples (living or freeze-dried samples from EF ponds, dried natural samples and one commercial pill). The data confirmed the existence of Clusters I and II, but also subdivided each of them into two Subclusters (A and B). In three cultures, simultaneous presence of types II.A and II.B was detected. It is likely that sequences of both types are contained in different copies of the ITS and that the three cultures represent cryptic duplicates of one unique genotype. The strains cultivated in the EF ponds belong to types I.A, II.A and II.B, while the winter ponds samples were a mixture of types I and II. Though there was surprisingly little sequence variability in the ITS sequences, we designed PCR primers which are specific for the two clusters (44 different positions) and for the four subclusters (2 to 4 different positions). [less ▲]

33 Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs

in Systematic & Applied Microbiology (1999), 22

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera ... [more ▼]

The level of genotypic relationship between czc+ soil bacteria mainly resistant to zinc (but also to various other metals), and related facultative hydrogenotrophs previously assigned to the genera Alcaligenes, Ralstonia, and Burkholderia was evaluated using ARDRA (Amplified Ribosomal DNA Restriction Analysis). The analysis included 44 strains isolated from harsh industrial environments in sediments, soils and wastes with high content of heavy metals. These strains were selected by their ability to grow in the presence of high concentrations of multiple heavy metals and to hybridise with czc or ncc probes. The czc operon confers resistance to cadmium, zinc and cobalt in strain Ralstonia eutropha CH34. The net operon confers resistance to nickel, cobalt and cadmium in strain 31A known as Alcaligenes xylosoxidans. The analysis showed a close phylogenetic clustering of the czc+ strains inside the Ralstonia genus despite of their different origins and that the Ralstonia genus contained also the hydrogenotrophs and some catabolic strains assigned to the genus Ralstonia eutropha, strains up to now registrated as CDC IV c-2 strains as well as reference strains belonging to Ralstonia solanacearum and Ralstonia pickettii. The ncc+ strains are phylogenetically less related to each other compared to the czc+ strains. This suggests that the tested czc+ strains and some of the ncc+ strains may be considered as belonging to the genus Ralstonia. Inside this major Ralstonia cluster, a subcluster gathers most of the czc+ isolates maybe giving a clue to define a new species. Besides, from 30 tested strains, 15 metal resistant strains of this subcluster proved to display the unusual mutator phenotype characteristic of the representative strain CH34 [less ▲]

Phylogenetic Relationships of Nonaxenic Filamentous Cyanobacterial Strains Based on 16S rRNA Sequence Analysis

in Journal of Molecular Evolution (1996), 42

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish ... [more ▼]

In order to determine the nearly complete 16S rRNA gene sequences of cyanobacteria originating from nonaxenic cultures, a cyanobacterium-specific oligonucleotide probe was developed to distinguish polymerase chain reaction (PCR) products of the cyanobacterial rRNA operons from those resulting from amplification of contaminating bacteria. Using this screening method the 16S rRNA genes of four nonaxenic filamentous cyanobacterial strains belonging to the genera Leptolyngbya and OscitIatoria were cloned and sequenced. For the genus Leptolyngbya, the 16S rRNA sequence of the axenic strain PCC 73110 was also determined. Phylogenetic trees were constructed based on complete and partial sequences. The results show that the strains Leptolyngbya Jbveolarum Kom~rek 1964/112, Leptolyngbya sp. VRUC 135 Albertano t985/1, and LeptoIyngbya boryanum PCC 73110 belong to the same cluster. Strain Oscillatoria cf. corallinae SAG 8.92, which contains the rare photosynthetic pigment CUphycoerythrin, is not closely related to other CUphycoerythrin- containing cyanobacteria. Oscillatoria agardhii CYA 18, which is a representative of planktonic Oscillatoria species that form toxic blooms in Norwegian inland waters, has no close relatives in the tree. [less ▲]

Molecular Evolution and Taxonomy of the Cyanobacteria

in Bryant, Donald A (Ed.) The Molecular Biology of Cyanobacteria (1994)

Molecular biology has provided new tools to decipher genetic information and can be used in attempts to reconstruct the evolution of organisms and improve their taxonomy. In the cyanobacteria, the use of ... [more ▼]

Molecular biology has provided new tools to decipher genetic information and can be used in attempts to reconstruct the evolution of organisms and improve their taxonomy. In the cyanobacteria, the use of molecular methods to study the genotypic relationships is underway, and initial results are promising. Different chemotaxonomic and macromolecular techniques are reviewed and their usefulness is evaluated. The most complete phylogenetic scheme of the cyanobacteria which is presently available is based on 16S rRNA sequence analysis. With this method, controversial taxonomic problems have been solved, such as the relationships among Pseudanabaena sp. strains or between the genera Arthrospira and Spirulina. In other cases, additional 16S rRNA sequences are necessary to obtain a clear picture. In addition to the cultivated strains, molecular ecology studies have contributed to the determination of new 16S rRNA sequence types, that have been retrieved directly from natural populations. The corresponding morphologies are presently unknown but may be revealed by the use of labeled probes annealing to specific 16S rRNA regions. For taxonomic purposes, it is necessary to find morphological features and simple testing methods which are congruent with the genotypic groupings. This information may be used to evaluate and revise existing classifications. The first stage in the development of such a polyphasic taxonomy is now underway. [less ▲]

Plasmids for heavy metal resistance in Alcaligenes eutrophus CH34: Mechanisms and applications

in FEMS Microbiology Reviews (1994), 14

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy ... [more ▼]

Alcaligenes eutrophus CH34 is the main representative of a group of strongly related strains (mostly facultative chemolithotrophs) that are well adapted to environments containing high levels of heavy metals. It harbors the megaplasmids pMOL28 and pMOL30 which carry resistance determinants to Co2+, Ni2+, CrO42-, Hg2+, Tl+, Cd2+, Cu2+ and Zn2+. Among the best characterized determinants are the cnr operon (resistance to Co, Ni) an pMOL28 and the czc operon on pMOL30 (resistance to Co, Cd and Zn). Although the two systems reveal a significant degree of amino acid similarity in the structural genes, the regulation of the operons is different. The resistance mechanism in both cases is based on efflux. The efflux mechanism leads to a pH increase outside of the cytoplasmic membrane. Metals are sequestered from the external medium through the bioprecipitation of metal carbonates formed in the saturated zone around the cell. This latter phenomenon can be exploited in bioreactors designed to remove metals from effluents. The bacteria are immobilized on composite membranes in a continuous tubular membrane reactor (CTMR). The effluent continuously circulates through the intertubular space, while the external surface of the tubes is in contact with the growth medium. Metal crystals are eventually removed by the effluent stream and collected on a glass bead column. The system has been applied to effluents containing Cd, Zn, Co, Ni and Cu. By introducing catabolic plasmids involved in the aerobic degradation of PCBs and 2,4-D into metal-resistant A. eutrophus strains, the application range was widened to include effluents polluted with both organic and inorganic substances. Biosensors have been developed which are based on the fusion of genes induced by metals to a reporter system, the lux operon of Vibrio fischeri. Bacterial luciferases produce light through the oxidation of fatty aldehydes. The gene fusions are useful both for the study of regulatory genes and for the determination of heavy metal concentrations in the environment. [less ▲]

Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA sequence analysis

in Systematic & Applied Microbiology (1993), 16

The primary structure of the small ribosomal subunit RNA (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The ... [more ▼]

The primary structure of the small ribosomal subunit RNA (SSU rRNA) was determined for 13 species belonging to 10 ascomycete families and for the basidiomycetous anamorphic yeast Rhodotorula glutinis. The sequences were fitted into an alignment of all hitherto published complete or nearly complete eukaryotic small subunit rRNA sequences. The evolutionary relationships within the fungi were examined by construction of a tree from 87 SSU rRNA sequences, corresponding to 71 different species, by means of a distance matrix method and bootstrap analysis. It confirms the early divergence of the zygomycetes and the classical division of the higher fungi into basidiomycetes and ascomycetes. The basidiomycetes are divided into true basidiomycetes and ustomycetes. Within the ascomycetes, the major subdivisions hemiascomycetes and euascomycetes can be recognized. However, Schizosaccharomyces pombe does not belong to the cluster of the hemiascomycetes, to which it is assigned in classical taxonomic schemes, but forms a distinct lineage. Among the euascomycetes, the plectomycetes and the pyrenomycetes can be distinguished. Within the hemiascomycetes, the polyphyly of genera like Pichia or Candida and of families like the Dipodascaceae and the Saccharomycetaceae can be observed. [less ▲]

Proceedings of the Symposium "Macromolecular identification and classification of organisms" (University of Antwerp, 7 December 1991): Introduction

in Belgian Journal of Botany (1992), 125

TAXONOMIC STUDY OF MARINE OSCILLATORIACEAN STRAINS (CYANOBACTERIA) WITH NARROW TRICHOMES. II. NUCLEOTIDE SEQUENCE ANALYSIS OF THE 16S RIBOSOMAL RNA

in Journal of Phycology (1992), 28

Partial 16S ribosomal RNA sequences from five marine oscillatoriacean strains with narrow trichomes were determined by a dideoxynucleotide-termination method. A phenogram was constructed by a distance ... [more ▼]

Partial 16S ribosomal RNA sequences from five marine oscillatoriacean strains with narrow trichomes were determined by a dideoxynucleotide-termination method. A phenogram was constructed by a distance matrix method including a bootstrap analysis. In addition, a consensus tree was built using cladistic analysis. The results were largely congruent and indicate that the five strains belong to two different lineages. The first lineage groups four phycoerythrin-producing strains with the strain PCC7375 (“Phormidium ectocarpi Gomont”). The second cluster groups strain PCC7105 (“Oscillatoria williamsii Drouet”) with the previously studied strain Microcoleus 10mfx. Comparisons to morphological data are made and the taxonomic level of the separations is estimated. [less ▲]

TAXONOMIC STUDY OF MARINE OSCILLATORIACEAN STRAINS (CYANOPHYCEAE, CYANOBACTERIA) WITH NARROW TRICHOMES .1. MORPHOLOGICAL VARIABILITY AND AUTECOLOGICAL FEATURES

in Archiv für Hydrobiologie (1991), 92

Twenty marine and one freshwater strain, belonging to the genera Oscillatoria, Phormidium, Plectonema and Spirulina (sensu GEITLER), exhibiting a cell width less than 3-mu-m were cultivated in different ... [more ▼]

Twenty marine and one freshwater strain, belonging to the genera Oscillatoria, Phormidium, Plectonema and Spirulina (sensu GEITLER), exhibiting a cell width less than 3-mu-m were cultivated in different light intensities, temperatures and salinities. The study of the morphological variability allows one to determine the stable and variable morphological characters. The tolerances to the culture conditions, the possibility of survival in unfavourable conditions and the capacity of chromatic adaptation were investigated. The autecological characters are discussed in relation to the original habitat as well as to the taxonomic grouping of the strains based on the morphology. [less ▲]

Growth and morphological variability of six strains of Phormidium cf. ectocarpi Gomont (Cyanophyceae) cultivated under different temperatures and light intensities

in Archiv fur Hydrobiologie, Suppl. Algological Studies (1988), 80